Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer–Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Abstract

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMO Xeno ) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMO Acineto ) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g DCW –1 at an optical density of OD 750 = 10) and an initial rate of 3.7 ± 0.2 mM h –1 . While the cells containing CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h –1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygen-dependent enzymatic processes.

Light-driven hydroxylation of testosterone by Synechocystis sp. PCC 6803 expressing the heterologous CYP450 monooxygenase CYP110D1

“…Cyanobacteria have a great potential to be used as whole-cell biocatalysts for the light-driven oxidation [1][2][3][4] or reduction of a variety of substrates, with activities close to what is required by industry. [5][6][7][8] Therefore, cyanobacteria are an appealing solution to perform chemical reactions more sustainably, helping to mitigate the impact that chemical and pharmaceutical industries have on the environment.…”

Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion

“…PCC 6803 were challenged with 12 a , in this case leading to a loss of product [26] . The problem was later overcome by using a faster BVMO [28] …”

“…[21,22] The photosynthetic machinery of the cyanobacterial host liberated electrons from water, ultimately producing intracellular NADPH, which in turn was used by the recombinant ERED to perform the biocatalytic reduction of C=C-bonds. Recently, this approach has been extended to other biocatalysts, including alcohol dehydrogenases (ADHs), [23,24] imine reductases (IREDs), [25] monooxygenases, [26][27][28] the AlkBGT hydroxylation-system, [29] and carboxylic acid reductases. [30] However, all of these systems report a range of shortcomings which prevent them from broad application and fast implementation, the most prominent being the challenges associated with cloning and expression of the target enzyme in the applied photoautotrophic organisms.…”

Recent Advances in Cyanobacterial Biotransformations

“…Unicellular strains like Synechocystis and Synechococcus elongatus PCC 7942 (from now on S. elongatus) were intensively studied as host for biotransformations in wild-type cells [19][20][21][22][23][24][25][26][27] and recombinant cells [17,[28][29][30][31][32][33][34][35][36][37]. The ease of handling, well elucidated genetic background, simple morphology, and possible genetic modification made both strains model strains over the years.…”

“…Time samples of the reaction can be analyzed to monitor the reaction curve and to calculate specific activity and conversion. Two strategies can be followed to genetically modify Synechocystis and S. elongatus: (1) genome integration via homologous recombination with subsequent segregation [17,[28][29][30][33][34][35][36] and (2) self-replicative vectors [37] Chem. Ing.…”